1. Field of the Invention
The invention relates to enzymatic RNA molecules which cleave mutant N-ras mRNA, preferably at a NUX cleavage site (N=any base, X=A, C or U), pharmaceuticals containing such molecules and use of such molecules for the preparation of pharmaceuticals for the treatment of diseases involving abnormal cell growth and/or differentiation.
2. Related Art
The growth and differentiation of cells depends on a variety of parameters and signal pathways. The inhibition of expression of certain signal transduction proteins may lead to an efficient therapy. Three ras-genes (Ha-ras, N-ras, Ki-ras) coding for ras-proteins are essentially involved in cell signal transduction and are members of the super gene family of small GTP/GDP binding proteins.
Studies of ras-oncogenes in tumors revealed point mutations leading to amino acid substitutions. Point mutations in the codons 12, 13, 59 and 61 cause structural changes of the GTP binding site and a reduced GTPase activity. Ras mutations have been detected in a wide variety of tumors such as pancreatic carcinomas, tumors in stomach and breast. Bos, Cancer Research 49:4682 (1989). N-ras mutations have been found in neuroblastoma, melanoma, acute myeloblastic leukaemia (AML), chronic myelogenous leukaemia (CML) and multiple myeloma. Portier et al., Oncogene 7:2539 (1992).
Enzymatic RNA molecules include Group I- and Group II-introns: Hammerhead ribozymes, hairpin ribozymes, hepatitis delta virus ribozymes ("axehead"), self-splicing introns and the subunit of RNAse P. Ribozymes of the hammerhead type are the smallest catalytic RNA ever found. Hammerhead ribozymes consist of three stems and 11 of 13 conserved nucleotides. They contain a consensus sequence which is part of the hammerhead structure. They recognize substrates containing NUX base triplet (N can be any base; X can be A, C, or U) and cleave the phosphodiester bond on the 3' side of X in trans position specifically. The GUC base triplet is cleaved most efficiently. Dahm et al., Biochemistry 30:9464 (1991).
Enzymatic RNA molecules which specifically cleave mRNA have been described previously (WO93/23057). Although enzymatic cleavage of ras mRNA has been mentioned in this application, it has not been disclosed which type of ras gene (Ha-ras, Ki-ras or N-ras) can be cleaved, and more importantly, it has not been shown which region within the ras mRNA sequence could be cleaved. Furthermore, only wild-type ras gene has been suggested as target, but no mutant ras genes.
In another application (WO 91/18913), ribozymes which cleave Ha-ras mRNA at codon 12 were described. However, the described ribozymes would not allow cleavage of other ras types such as N-ras. The described ribozymes are also not stable under in vivo conditions.
Therefore, enzymatic RNA molecules that cleave mutant N-ras mRNA have potential as therapeutic agents, but a need exists for ribozymes that cleave mutant N-ras mRNA in predictable manner.